Identification and Phylogenetic Analysis of the R2R3-MYB Subfamily in Brassica napus

The R2R3-MYB sub-family proteins are composed of most members of MYB (v-Myb avian myeloblastosis viral oncogene homolog) protein, a plant-specific transcription factor (TF) that is classified into four classes depending on the number of MYB repeats. R2R3-MYB TFs are involved in physiological and biochemical processes. However, the functions of the Brassica napus R2R3-MYB genes are still mainly unknown. In this study, 35 Brassica napus MYB (BnaMYB) genes were screened in the genome of Brassica napus, and details about their physical and chemical characteristics, evolutionary relationships, chromosome locations, gene structures, three-dimensional protein structures, cis-acting promoter elements, and gene duplications were uncovered. The BnaMYB genes have undergone segmental duplications and positive selection pressure, according to evolutionary studies. The same subfamilies have similar intron–exon patterns and motifs, according to the genes’ structure and conserved motifs. Additionally, through cis-element analysis, many drought-responsive and other stress-responsive cis-elements have been found in the promoter regions of the BnaMYB genes. The expression of the BnaMYB gene displays a variety of tissue-specific patterns. Ten lignin-related genes were chosen for drought treatment. Our research screened four genes that showed significant upregulation under drought stress, and thus may be important drought-responsive genes. The findings lay a new foundation for understanding the complex mechanisms of BnaMYB in multiple developmental stages and pathways related to drought stress in rapeseed.


Introduction
In land plants, drought is often a major stress that can reduce crop productivity severely [1]. Rapeseed plants suffer terrible consequences when there is a water shortage, resulting in changed physiological processes (photosynthesis rates, osmotic protection, and oil contents) and reduced plant growth [2]. Rapeseed is quite vulnerable to drought stress during the flowering stage, and it has been projected that global climatic oscillations, which are the cause of severe and prolonged drought in some areas of the biosphere, will reduce rapeseed output [3]. Therefore, finding the methods that can mitigate drought stress is critical for B. napus (Brassica napus L.). A method of accumulating secondary metabolites are formed through accumulating lignin [4][5][6][7][8][9]. In poplar (Populus), overexpression of the transcription factor PdNF-YB21 promotes root growth, along with drought tolerance [9]. Furthermore, overexpression of VlbZIP30 in grapevine (Vitis vinifera) [8], poCCoAOMT in tobacco (Nicotiana benthamiana) [10], IbLEA14 in sweet potato (Ipomoea batatas) [11], osTF1L in rice [12], and PeLAC10, CmCAD2 and CmCAD2 in Arabidopsis (Arabidopsis thaliana) has been found [7,12,13]. All these genes enhance the synthesis of lignin and drought resistance. Inbred lines of maize (Zea mays) that are more lignified than drought-sensitive lines imply that lignification is a key mechanism for drought stress adaptation [14].
There are several genes within the MYB gene family that act as TFs for plants. There is a conserved DBD (DNA-binding domain) in the MYB transcription factor, which can be composed of up to four imperfect repeats, known as R repeats [15,16]. Each R repeat's second and third helices join with specific gene-specific promoter sequences to create a helixturn-helix (HTH) structure, which is 50-55 amino acids long [15,17]. In addition, the third αhelix is usually responsible for recognizing short DNA sequences [18]. Sequence similarity determines whether the MYB domain repeats belong to the R1, R2, or R3 category [16,19]. Members of the plant MYB superfamily generally consist of one to four imperfect MYB repeats, and there are four types of repetitions: 1R-MYB (one or two separated repeats), 2R-MYB (R2R3-MYB, two adjacent repeats), 3R-MYB (three adjacent repeats), and 4R-MYB (consisting of four adjacent repeats) [16]. The R2R3-MYB family has considerably grown in size and now dominates in the plant lineage [20].
In a recent study [21], 2120 R2R3-MYB genes were identified in six brassica plants, including 130 in Arabidopsis, 236 in B. rapa, 247 in B. oleracea, 248 in B. nigra, 425 in B. napus, 422 in B. juncea and 412 in B. carinata. However, the number of R2R3-MYBs in Arabidopsis, B. rapa and B. napus were different from those found in previous studies, namely, 126 in Arabidopsis [18], 256 in B. rapa [22], and 249 in B. napus [23]. The main reason may be that with the development of third-generation sequencing technology, the information has become more complete.
R2R3-MYB TFs have been shown to synthesize hormones, participate in signal transduction, and be involved in physiological and biochemical processes, particularly in reaction to a variety of biotic and abiotic stimuli [16,[24][25][26]. In addition, R2R3-MYB also contributes significantly to lignin synthesis [16]. The R2R3-MYB transcription factor PbrMYB169 protein of pear (Pyrus bretschneideri) significantly activated the promoters of lignin genes C3H1, CCR1, CCOMT2, CAD, 4CL1, 4CL2, HCT2 and LAC18 by binding with the AC element in the promoter, thus positively regulating the lignification in the cell matrix of pear fruit [27]. The constitutive expression of three members of MYB transcription factor family (PtrMYB92, PtrMYB3 and PtrMYB20) in poplar activated the expression of lignin biosynthesis gene and induced the ectopic deposition of lignin. At the same time, they can also activate the promoter of poplar wood biosynthesis gene [28,29]. The overexpression of TaMYB4 leads to the reduction of the transcription of CAD and CCR genes involved in the lignin biosynthesis pathway, which has a negative regulatory effect on the lignin biosynthesis in wheat (Triticum aestivum L.) [30]. ZmMYB31 and ZmMYB42 can downregulate the COMT genes of maize (Zea mays L.) and Arabidopsis, and overexpression results in a reduction of lignin content in transgenic plants [31]. The overexpression of MusaMYB31 can downregulate many common phenylpropane genes and lignin biosynthesis pathways, and lignin biosynthesis is negatively regulated in banana (Musa acuminata) [32]. According to these results, we found that the R2R3-MYB transcription factor can bind to the promoter and significantly activate the lignin gene. However, the expression of some R2R3-MYB transcription factors can downregulate the genes in the lignin synthesis pathway, thus negatively regulating the lignin biosynthesis in plants. We have learned that lignin deposition has a great impact on drought responses, so R2R3-MYB is responsive to drought stress while regulating lignin synthesis.
In Arabidopsis, there are about 126 members of the R2R3-MYB subfamily, which is roughly divided into 25 subgroups and distributed across 11 branches [18]. The functions of most members have been identified, and they have been found to be involved in the control of plant-specific processes, including (i) primary and secondary metabolism, (ii) cell fate and identity, (iii) development processes, and (iv) the response to biological and abiotic stresses. We found in the literature that AtMYB26 can control the deposition of secondary walls in the anthers [33]. AtMYB46 is a positive regulator of lignin biosynthesis in fibers and vessels [34]. These two genes may be able to cope with drought stress by regulating the production of lignin. These two genes are not included in the 25 subgroups but are in the same branch as Subgroups 13 and 16. Therefore, we preliminarily selected the Brassica napus homologs to this branch for further analysis.
In this work, 35 BnMYB genes of rapeseed were discovered by comparing the genomic sequences of the S13 and S16 subfamilies of the known R2R3-MYB from Arabidopsis thaliana. Additionally, we used the method of bioinformatic analysis to effectively process, analyze, and visualize a large amount of data from the field of life science. The physical and chemical characteristics of the BnMYB genes' cis-acting promoter and their gene duplication were clarified, as well as their evolutionary relationships, genomic positions, gene structures, and three-dimensional membrane proteins. We also looked at how the BnaMYB gene family was expressed in various tissues and how they responded to drought treatment. Further research into the functional characterization of BnMYBs may benefit from our findings.

Identification of the R2R3-MYB Transcription Factor Family in Rapeseed
To identify R2R3-MYB family genes in rapeseed, 12 AtTCP protein sequences were used as queries for a BlastP search against the rapeseed genome. As a result, 35 BnMYB genes were consistent with the R2R3-MYB domain (Table 1). Hereafter, these genes are named BnMYB1 to BnMYB35. The physical and chemical characteristics of each member were then examined and predicted. The proteins that the BnaMYB genes encoded ranged in size from 239 amino acids (BnaMYB35) to 370 amino acids (BnaMYB17, BnaMYB18); the MW ranged from 77,433.37 kDa (BnaMYB8) to 195,053.44 kDa (BnaMYB23); and the PI ranged from 4.89 (BnaMYB23) to 5.11 (BnaMYB8). The predicted subcellular location of each BnaMYB protein was in the nucleus. Table 1 also contains the length of the coding sequences.

Location of the Chromosomes and Phylogenetic Analysis
Additionally, the 35 BnaMYB genes were found on the chromosomes ( Figure 1). The rapeseed genome had 35 R2R3-MYB genes that were widely distributed throughout 19 chromosomes. On chromosome A10, there was just one gene; on chromosomes A01, A02, C02, C03, C07 and C09, there were two genes; on chromosomes A05, A06, and C01, there were three; and on chromosomes A03, A09 and C05, there were four. Table S1 shows the position of each gene on the chromosomes. We built a phylogenetic tree using 133 MYB proteins from seven different species, including Arabidopsis, Brassica napus, Brassica oleracea, Brassica rapa, Capsella rubella, Oryza sativa, Raphanus sativus and Zea mays, to better understand the evolutionary relationships among the MYB genes of the different species. Table S2 shows the protein sequences. These MYB genes were divided into three groups, as seen in Figure 2A. The majority of MYB members were found in the third group, which consisted of seven species, while the second group had the fewest MYB genes. Groups A, B, and C contained 10, 5 and 20 BnaMYB gene members, respectively (marked with asterisks in Figure 2A). Members of BnaMYB that clustered together may have a special connection and carry out similar functions. We built a phylogenetic tree using 133 MYB proteins from seven different species, including Arabidopsis, Brassica napus, Brassica oleracea, Brassica rapa, Capsella rubella, Oryza sativa, Raphanus sativus and Zea mays, to better understand the evolutionary relationships among the MYB genes of the different species. Table S2 shows the protein sequences. These MYB genes were divided into three groups, as seen in Figure 2A. The majority of MYB members were found in the third group, which consisted of seven species, while the second group had the fewest MYB genes. Groups A, B, and C contained 10, 5 and 20 BnaMYB gene members, respectively (marked with asterisks in Figure 2A). Members of BnaMYB that clustered together may have a special connection and carry out similar functions. The number of R2R3-MYB genes in each subspecies within each group was then tallied ( Figure 2B). Although the first group had no examples of ZmMYB, the seven species were distributed throughout the three groups. Each group of species had two to four copies of the R2R3-MYB genes, depending on the quantity of these genes in A. thaliana. We found that the BnaMYB genes in Group A were all homologs of AtMYB46, and the BnaMYB genes in Group C were all homologs of AtMYB86.

Conserved Motifs and Gene Structure
Because Arabidopsis is a model plant, the research on its genome and gene function is the most complete. Rape and Arabidopsis belong to the same family of Cruciferae, with The number of R2R3-MYB genes in each subspecies within each group was then tallied ( Figure 2B). Although the first group had no examples of ZmMYB, the seven species were distributed throughout the three groups. Each group of species had two to four copies of the R2R3-MYB genes, depending on the quantity of these genes in A. thaliana. We found that the BnaMYB genes in Group A were all homologs of AtMYB46, and the BnaMYB genes in Group C were all homologs of AtMYB86.

Conserved Motifs and Gene Structure
Because Arabidopsis is a model plant, the research on its genome and gene function is the most complete. Rape and Arabidopsis belong to the same family of Cruciferae, with the closest genetic relationships and the clearest comparisons of genome homology. Therefore, we created an evolutionary tree with 35 BnaMYB protein sequences from B. napus and 12 AtMYB protein sequences from A. thaliana to examine the structural variety of BnaMYB genes. All of the R2R3-MYB genes were divided into Groups I, II and III ( Figure 3A). Their conserved protein domains and gene structure were then investigated further.  The full-length protein sequences were examined to identify their conserved motifs ( Figure 3B). The R2R3-MYB genes' conserved motifs ranged from two to five. Ten conserved motifs in all were found; their widths ranged from 26 to 141 amino acids (Table  S3). In addition, individuals of the same group had common conserved motifs. For instance, Motifs 7 and 9 were particular to Class I, Motif 8 to Class II and Motifs 4, 5 and 10 to Class III. The quantity and organization of the MYB motifs in the various groups, however, varied slightly. In Group II, BnaMYB9 had fewer conserved motifs than other members, whilst BnaMYB17, BnaMYB18 and AtMYB103 all had conserved motifs that were different from the other members.
To further understand the development of the R2R3-MYB gene family in rape, according to the findings, there were between one and either two or three exons and introns, respectively ( Figure 3C). Sixteen genes had two exons and one intron, whereas 31 genes had three exons and two introns. Exon gains and losses were discovered during the evolution of the MYB family of genes. Our findings suggested that over the evolution of the rapeseed genome, the exon-intron architecture of MYB genes remained relatively stable. Additionally, the BnaMYB gene members within a class had gene architectures that were quite similar and consistent with their evolutionary groups. In summary, according to our The full-length protein sequences were examined to identify their conserved motifs ( Figure 3B). The R2R3-MYB genes' conserved motifs ranged from two to five. Ten conserved motifs in all were found; their widths ranged from 26 to 141 amino acids (Table S3). In addition, individuals of the same group had common conserved motifs. For instance, Motifs 7 and 9 were particular to Class I, Motif 8 to Class II and Motifs 4, 5 and 10 to Class III. The quantity and organization of the MYB motifs in the various groups, however, varied slightly. In Group II, BnaMYB9 had fewer conserved motifs than other members, whilst BnaMYB17, BnaMYB18 and AtMYB103 all had conserved motifs that were different from the other members.
To further understand the development of the R2R3-MYB gene family in rape, according to the findings, there were between one and either two or three exons and introns, respectively ( Figure 3C). Sixteen genes had two exons and one intron, whereas 31 genes had three exons and two introns. Exon gains and losses were discovered during the evolution of the MYB family of genes. Our findings suggested that over the evolution of the rapeseed genome, the exon-intron architecture of MYB genes remained relatively stable. Additionally, the BnaMYB gene members within a class had gene architectures that were quite similar and consistent with their evolutionary groups. In summary, according to our analysis of the conserved motif composition, gene structures, and phylogenetic interactions, the consistency of the class organization was convincingly sustained, showing that the MYB proteins have extraordinarily well-maintained amino acid residues and that the members within a class may play parallel roles.

Multi-Sequence Alignment and Predicted 3d Protein Structures
We used 3D structure prediction analysis and multiple sequence alignment to clarify the structural properties of the BnaMYB proteins ( Figures S1 and S2). These findings led us to the conclusion that the MYB DNA-binding domains in these proteins are highly conserved. The BnaMYB proteins were split into three groups on the basis of their genetic compatibility.

Collinearity within B. napus and among Different Species
Analysis of genome-wide replication is crucial for understanding the genesis, evolution, and genome-wide expansion of organisms. To further understand the reasons behind gene replication events in BnaMYB, we therefore examined the replication events of the R2R3-MYB gene family in B. napus. A04, A07, A08, C04, C06 and C08 were the only chromosomes without fragment repeats, along with 48 pairs of genes with large fragment repeats ( Figure 4). These findings suggested that the amplification and evolution of the R2R3-MYB genes in the B. napus genome may have been significantly influenced by the replication of large fragments. analysis of the conserved motif composition, gene structures, and phylogenetic interactions, the consistency of the class organization was convincingly sustained, showing that the MYB proteins have extraordinarily well-maintained amino acid residues and that the members within a class may play parallel roles.

Multi-Sequence Alignment and Predicted 3d Protein Structures
We used 3D structure prediction analysis and multiple sequence alignment to clarify the structural properties of the BnaMYB proteins ( Figures S1 and S2). These findings led us to the conclusion that the MYB DNA-binding domains in these proteins are highly conserved. The BnaMYB proteins were split into three groups on the basis of their genetic compatibility.

Collinearity within B. napus and among Different Species
Analysis of genome-wide replication is crucial for understanding the genesis, evolution, and genome-wide expansion of organisms. To further understand the reasons behind gene replication events in BnaMYB, we therefore examined the replication events of the R2R3-MYB gene family in B. napus. A04, A07, A08, C04, C06 and C08 were the only chromosomes without fragment repeats, along with 48 pairs of genes with large fragment repeats ( Figure 4). These findings suggested that the amplification and evolution of the R2R3-MYB genes in the B. napus genome may have been significantly influenced by the replication of large fragments. The evolution of the R2R3-MYB gene family in Brassica has been tracked in previous research [21]. In order to specifically understand the evolutionary relationship of target genes, we analyzed the homologous relationship among Arabidopsis, B. napus (A and C subgenomes), B. rapa (A genome), and B. oleracea (C genome) ( Figure 5). The collinearity analysis of MYB showed that there were a large number of orthologous MYB genes in Arabidopsis, B. rapa, B. oleracea, and B. napus. There were 16 pairs of genes in Arabidopsis The evolution of the R2R3-MYB gene family in Brassica has been tracked in previous research [21]. In order to specifically understand the evolutionary relationship of target genes, we analyzed the homologous relationship among Arabidopsis, B. napus (A and C subgenomes), B. rapa (A genome), and B. oleracea (C genome) ( Figure 5). The collinearity analysis of MYB showed that there were a large number of orthologous MYB genes in Arabidopsis, B. rapa, B. oleracea, and B. napus. There were 16 pairs of genes in Arabidopsis and B. rapa that showed collinearity, and 15 B. rapa R2R3-MYB genes had homologous genes in Arabidopsis, 10 of which were multi-copy genes and 5 were single-copy genes. B. rapa lacked AtMYB50 homologs, indicating that gene loss occurred in B. rapa during evolution. Furthermore, 16 B. oleracea R2R3-MYB genes contained similar genes to Arabidopsis. The A and C subgenomes of B. napus mostly overlapped those of the related diploids B. rapa and B. oleracea. Fourteen similar gene pairs were discovered in the A genome of B. napus and B. rapa, and 21 in the C genome of B. napus and B. oleracea. Even if gene loss has occurred, the great majority of R2R3-MYB genes in B. napus are still present and have contributed to the evolution of the MYB gene family. and B. rapa that showed collinearity, and 15 B. rapa R2R3-MYB genes had homologous genes in Arabidopsis, 10 of which were multi-copy genes and 5 were single-copy genes. B. rapa lacked AtMYB50 homologs, indicating that gene loss occurred in B. rapa during evolution. Furthermore, 16 B. oleracea R2R3-MYB genes contained similar genes to Arabidopsis.

Synteny of BnMYB Genes
We compared the CDS sequences ( Figure S3), which showed the similarity and identity of these sequences more clearly. We estimated the synonymous (Ks) and nonsynonymous (Ka) replacement rates (Ka/Ks) of 44 segmented repeated pairs (Table S4). The average Ka/Ks ratio of fragmented repeated gene pairs was 0.61. The calculation results showed that the Ka/Ks ratio of all duplicate BnMYB gene pairs was less than 1, which means that they are undergoing a purification selection process.

Cis-Acting Elements and Functional Annotation
Transcriptional regulators bind to the cis-acting elements, which control gene transcription. We examined the cis-acting elements of the 1500 bp upstream sequence of BnaMYB promoters in order to determine the potential function of the BnaMYB genes, excluding components with uncertain functions and general transcriptional regulatory elements (Table 2, Figure 6). All details of cis elements of each gene are listed in Supplementary Table S5. ABRE, the AuxRR core, a TGA element, a TGA box, a TGACG motif, a CGTCA motif, a P box, a GARE motif, TCA and a TCA element were distinguished as being responsive elements related to four phytohormones (abscisic acid (ABA), auxin, methyl jasmonate (MeJA), gibberellin (GA), and salicylic acid (SA)). Overall, we focused on and identified eight types. We also found four stress-related (drought, low-temperature, anaerobic, defense, and stress) responsive elements, including MBS, MYB, MYC, LTR, ARE and TC-rich repeats, suggesting their involvement in stress responses. The drought stress-responsive elements MBS, MYC and MYB were primarily found in 13 genes, 31 genes and 35 genes, respectively. These findings revealed that the majority of BnaMYB genes have drought-responsive elements, indicating that BnaMYB genes may be extremely important in responding to the stress caused by drought.

Synteny of BnMYB Genes
We compared the CDS sequences ( Figure S3), which showed the similarity and identity of these sequences more clearly. We estimated the synonymous (Ks) and nonsynonymous (Ka) replacement rates (Ka/Ks) of 44 segmented repeated pairs (Table S4). The average Ka/Ks ratio of fragmented repeated gene pairs was 0.61. The calculation results showed that the Ka/Ks ratio of all duplicate BnMYB gene pairs was less than 1, which means that they are undergoing a purification selection process.

Cis-Acting Elements and Functional Annotation
Transcriptional regulators bind to the cis-acting elements, which control gene transcription. We examined the cis-acting elements of the 1500 bp upstream sequence of BnaMYB promoters in order to determine the potential function of the BnaMYB genes, excluding components with uncertain functions and general transcriptional regulatory elements (Table 2, Figure 6). All details of cis elements of each gene are listed in Supplementary Table S5. ABRE, the AuxRR core, a TGA element, a TGA box, a TGACG motif, a CGTCA motif, a P box, a GARE motif, TCA and a TCA element were distinguished as being responsive elements related to four phytohormones (abscisic acid (ABA), auxin, methyl jasmonate (MeJA), gibberellin (GA), and salicylic acid (SA)). Overall, we focused on and identified eight types. We also found four stress-related (drought, low-temperature, anaerobic, defense, and stress) responsive elements, including MBS, MYB, MYC, LTR, ARE and TC-rich repeats, suggesting their involvement in stress responses. The drought stress-responsive elements MBS, MYC and MYB were primarily found in 13 genes, 31 genes and 35 genes, respectively. These findings revealed that the majority of BnaMYB genes have drought-responsive elements, indicating that BnaMYB genes may be extremely important in responding to the stress caused by drought.

Tissue-Specific Expression Patterns of BnaMYB Genes
Through the use of RNA-seq data from Brassica napus (ZS11 variant) (BioProject ID: PRJCA001495), the histo-specific expression profiles of BnMYB genes were detected in nine different tissues and organs (i.e., stems, roots, siliques, cotyledons, sepals, seeds, buds, leaves, and petals). The expression patterns of all BnaMYB genes varied in the different tissues, as shown in Figure 7 and

Tissue-Specific Expression Patterns of BnaMYB Genes
Through the use of RNA-seq data from Brassica napus (ZS11 variant) (BioProject ID: PRJCA001495), the histo-specific expression profiles of BnMYB genes were detected in nine different tissues and organs (i.e., stems, roots, siliques, cotyledons, sepals, seeds, buds, leaves, and petals). The expression patterns of all BnaMYB genes varied in the different tissues, as shown in Figure 7 and Table S6. For instance, the majority of genes displayed greater expression levels in the stems, including BnaMYB3, BnaMYB5, BnaMYB7, BnaMYB11, BnaMYB14, BnaMYB15, BnaMYB16, BnaMYB17, BnaMYB18, BnaMYB19, BnaMYB23, BnaMYB25, BnaMYB26, BnaMYB27, BnaMYB28, BnaMYB30, BnaMYB31, BnaMYB33, BnaMYB34 and BnaMYB35. In the cotyledons, a few genes showed higher expression levels, including BnaMYB2, BnaMYB4, BnaMYB6, BnaMYB7, BnaMYB8, BnaMYB10, BnaMYB14, BnaMYB20, BnaMYB22, BnaMYB24, BnaMYB26, BnaMYB29, BnaMYB31 and BnaMYB32. In the roots, BnaMYB4, BnaMYB11, BnaMYB14, BnaMYB15, BnaMYB16, BnaMYB17, BnaMYB18, BnaMYB27, BnaMYB31 and BnaMYB33 displayed higher expression patterns. In the sepals, BnaMYB1, BnaMYB9, BnaMYB21 and BnaMYB28 exhibited higher expression patterns. In the siliques, BnaMYB5, BnaMYB15 and BnaMYB33 displayed higher expression patterns. Likewise, in the seeds, many genes, including BnaMYB12 and BnaMYB27, showed higher expression patterns. These findings led us to the conclusion that the BnaMYB gene family was essential for rapeseed's development at all stages, and that other members with similar expression traits might serve related purposes. BnaMYB33 displayed higher expression patterns. Likewise, in the seeds, many genes, including BnaMYB12 and BnaMYB27, showed higher expression patterns. These findings led us to the conclusion that the BnaMYB gene family was essential for rapeseed's development at all stages, and that other members with similar expression traits might serve related purposes.

Expression Profiles of BnMYB Genes under Drought Stress
The majority of the BnaMYB genes had drought-responsive elements, which suggests that they may be involved in the response to drought, according to an analysis of the cisacting elements in the upstream sequence of the BnaMYB promoters we conducted before. We chose 10 lignin-related genes (BnaMYB3, BnaMYB5, BnaMYB7, BnaMYB11,  BnaMYB19, BnaMYB23, BnaMYB25, BnaMYB26, BnaMYB30 and BnaMYB35) and submitted the 10 genes to drought treatment for 7 days to further discover their potential function (Figure 8). The primers used for gene expression analysis are shown in Table S7.

Expression Profiles of BnMYB Genes under Drought Stress
The majority of the BnaMYB genes had drought-responsive elements, which suggests that they may be involved in the response to drought, according to an analysis of the cis-acting elements in the upstream sequence of the BnaMYB promoters we conducted before. We chose 10 lignin-related genes (BnaMYB3, BnaMYB5, BnaMYB7, BnaMYB11, BnaMYB19, BnaMYB23, BnaMYB25, BnaMYB26, BnaMYB30 and BnaMYB35) and submitted the 10 genes to drought treatment for 7 days to further discover their potential function (Figure 8). The primers used for gene expression analysis are shown in Table S7. and BnaMYB30 were very low in the first 4 days, significantly increased on Days 5 and 6, and rapidly increased on Day 7 to reach the peak, indicating that drought treatment could activate the expression of these four genes.
The majority of BnaMYB genes generally had various drought expression patterns, indicating that these MYB genes were engaged in the pathway that causes drought stress. Four genes (BnaMYB7, BnaMYB11, BnaMYB26 and BnaMYB30) showed significantly upregulated expression under drought stress.

Discussion
The R2R3-MYB gene family of B. napus and related species was biologically analyzed in this work, including their geographic position, phylogenetic analysis, gene structure, homologous protein domains, and predicted 3D protein structure. The findings demonstrated that the 19 rapeseed chromosomes contained an unequal distribution of 35 BnaMYB family members. These individuals were divided into three groups through phylogenetic analysis, and the majority of the genes within each subgroup shared similar gene structures, conserved protein domains, and three-dimensional protein structures, which is indicative of the BnaMYB gene family's evolutionary stability.

Characterization and Evolution of the R2R3-MYB Gene Family in Rapeseed
Allotetraploid rapeseed is a crop that has been engaged in extensive genome replication and combination activities [35]. One of the biggest obstacles to agricultural production in the modern world is dry soils. Crop plants have been bred conventionally and genetically to increase tolerance or resistance to drought and salinity in an effort to boost agricultural productivity in impacted areas. Future breeding and genetic engineering were very low in the first 4 days, significantly increased on Days 5 and 6, and rapidly increased on Day 7 to reach the peak, indicating that drought treatment could activate the expression of these four genes.
The majority of BnaMYB genes generally had various drought expression patterns, indicating that these MYB genes were engaged in the pathway that causes drought stress. Four genes (BnaMYB7, BnaMYB11, BnaMYB26 and BnaMYB30) showed significantly upregulated expression under drought stress.

Discussion
The R2R3-MYB gene family of B. napus and related species was biologically analyzed in this work, including their geographic position, phylogenetic analysis, gene structure, homologous protein domains, and predicted 3D protein structure. The findings demonstrated that the 19 rapeseed chromosomes contained an unequal distribution of 35 BnaMYB family members. These individuals were divided into three groups through phylogenetic analysis, and the majority of the genes within each subgroup shared similar gene structures, conserved protein domains, and three-dimensional protein structures, which is indicative of the BnaMYB gene family's evolutionary stability.

Characterization and Evolution of the R2R3-MYB Gene Family in Rapeseed
Allotetraploid rapeseed is a crop that has been engaged in extensive genome replication and combination activities [35]. One of the biggest obstacles to agricultural production in the modern world is dry soils. Crop plants have been bred conventionally and genetically to increase tolerance or resistance to drought and salinity in an effort to boost agricultural productivity in impacted areas. Future breeding and genetic engineering plans are likely to be greatly aided by a knowledge of the genomic mechanisms underlying plants' responses to drought stress [36].
In plants, the MYB family has a significant function. The function of MYBs has been systematically researched in Arabidopsis [16,37], Setaria italica (Muthamilarasan et al., 2014), Vitis vinifera [38], Zea mays [25], Populus trichocarpa [39], Gossypium raimondii [40], and other plants, especially Arabidopsis and Oryza sativa [24]. The largest MYB gene subfamily in plants is the R2R3-MYB subfamily. Genetic methods have been used to characterize a large number of R2R3-MYB proteins, and it has been discovered that these proteins are involved in the regulation of a variety of plant-specific processes, such as (i) primary and secondary metabolism, (ii) cell fate and identity, (iii) developmental processes, and (iv) responses to biotic and abiotic stresses [16]. To the best of our knowledge, the rapeseed genome does not yet have a complete description of the R2R3-MYB gene family. The availability of rapeseed's genome sequences makes it possible to identify rapeseed genes across the entire genome [35]. Here, we identified 35 BnaMYB genes in the genome of rapeseed.
The BnaMYB gene family may have developed in the rapeseed genome through segmental events and many tandem duplications. A key strategy for gene expansion is tandem duplication. Genes that have been enlarged by tandem duplication are always dispersed throughout chromosomes as a group [41,42]. Exon-intron connections and numbers, in addition to duplication events, can help to explain how the gene family has evolved [43][44][45]. When the gene structures of BnaMYBs and members of the same group were studied, it was shown that MYB genes contributed equally to exon/intron allocation in terms of exon and intron numbers, while BnaMYBs showed similar motif patterns. These findings suggest that these R2R3-MYB members could play similar roles in a range of abiotic stressors.

Diversity in the Functional Expression of BnaMYB Genes in Brassica napus
We looked at the expression patterns of 35 BnaMYB genes in diverse organs using publicly accessible data. The results showed that they varied in their patterns of expression in different tissues and that they were expressed in different tissues. In stems and cotyledons, 85.7% of BnaMYB genes were primarily expressed, while the primary expression of 10 genes was detected in the roots. BnaMYB12 was only strongly expressed in the seeds and less so in other tissues. BnaMYB1, BnaMYB9, BnaMYB21 and BnaMYB28 displayed higher transcription levels in the sepals. These findings suggest that the BnaMYB gene family might well be important for rapeseed's development and expansion at all stages. Twenty of the BnaMYB genes were mostly expressed in the stems; therefore, they may play a crucial role in how the stems react to environmental influences. Little research has been conducted on Brassica napus, despite the fact that several R2R3-MYB genes have been linked to drought adaptations and endurance in some species, particularly in rice and Arabidopsis [46][47][48][49]. Hence, our study of BnaMYBs is helpful to analyze the response of R2R3-MYB to drought stress in Brassica napus.
Numerous members of the plant R2R3-MYB family have been identified as transcription factors that are triggered by abiotic stress to date [47,50,51]. Cotton's resilience to drought can be improved by transgenic Arabidopsis overexpressing the R2R3-MYB gene GaMYB85 [52]. Arabidopsis overexpressing LpMYB1 is more tolerant to salt and drought because of this R2R3-MYB factor [53]. Under prolonged drought stress, TaMYBsdu1 increases noticeably in the roots and leaves of wheat [47]. TaMYB33 improves tolerance to salt and drought, partly by restoring the osmotic equilibrium and eliminating superoxide radicals [54,55]. TaMYB30-B and TaMYB19-B can increase the ability of transgenic Arabidopsis plants to withstand drought stress [56,57]. These studies indicate that R2R3-MYB has the function of dealing with drought stress in wheat, cotton, and Arabidopsis and their function during drought stress is affected by the ABA synthesis and signal transduction pathway. For instance, the accumulation of 35S:GaMYB85 resulted in the transcription of RD22, RD29A, ADH1, AB15, and P5CS. The AB15 gene, which codes for a bZIP TF, is known to be an ABA-responsive gene that is important for seed germination under drought stress [58,59]. In light of the previous analysis, we speculated that drought stress in Brassica napus is affected by the lignin content. MYB46 controls not only the signaling molecules but also the genes that produce all three of the primary components of the secondary walls: grain, hemicellulose, and lignin [60]. Additionally, MYB85 controls the production of lignin by regulating the lignin-synthesizing genes and, when overexpressed, results in ectopic lignin deposition [61]. This indicates that the BnaMYB gene family may affect lignin synthesis, and lignin synthesis responds to drought stress. We will pay attention to this in future research to deeply understand the relationship between the BnaMYB gene family and lignin.
The tissue-specific expression patterns reflected which tissues of the plant the gene mainly acts on. We found that 20 genes were highly expressed mainly in the stem, indicating that these genes were probably involved in lignin synthesis. Ten of these genes were selected for drought treatment, and their expression profiles were detected under drought stress. Our RT-qPCR data showed that the BnaMYB gene had significant changes under drought conditions, which proved that 10 genes were responsive to drought stress. The expression of BnaMYB5 decreased significantly under drought stress, indicating that this gene could not cope with drought; BnaMYB3, BnaMYB19, BnaMYB23, BnaMYB25 and BnaMYB35 showed U-shaped curves, indicating that these five genes did not show drought resistance under mild drought stress but showed drought resistance under severe drought. Four genes (BnaMYB7, BnaMYB11, BnaMYB26 and BnaMYB30) were significantly upregulated under drought stress, indicating that they are probably drought-resistant genes. According to our understanding, this is the first evidence indicating that R2R3-MYB genes in B. napus are implicated in the response to drought stress. This work is helpful for understanding the characteristics and functions of R2R3-MYB genes in different species.

Chromosome Location and Phylogenetic Analysis
The BnPIR website (http://cbi.hzau.edu.cn/bnapus, accessed on 5 March 2022) provided information on the chromosome location of BnaMYBs. After that, the distribution status of the BnaMYBs discovered on the chromosomes was shown using MapChart software. With the aid of Adobe Illustrator, the outcomes were improved.
We  [62] was used to search for orthologous, co-orthologous, and paralogous genes in Arabidopsis thaliana, Brassica oleracea, Brassica rapa, Capsella rubella, Oryza sativa, Raphanus sativus and Zea mays using entire R2R3-MYB protein sequences. The E-value cut-off of an all-against-all BLASTP alignment process was set at 1 ×10 −10 , and the alignments with a match cut-off value lower than 50 were eliminated.
These plants' MYB protein sequences were analyzed using Clustal W after the protein sequences of MYB family members from B. napus, Arabidopsis, B. oleracea, B. rapa, Capsella rubella, Oryza sativa, Raphanus sativus and Zea mays were retrieved. Additionally, MEGA 11.0 software was used to create the evolutionary tree using the neighbor joining method (NJ) once the results of sequence alignment were obtained [63,64]. Finally, we used Evolview (http://www.omicsclass.com/article/671, accessed on 13 March 2022) to visualize the evolutionary tree.

Distinct Gene Structure and Conserved Protein Domain of R2R3-MYBs
The gene structure of the R2R3-MYB genes contains intron and exon information [65]. MEME Suite (https://meme-suite.org/meme/, accessed on 30 March 2022) was used to examine the homologous domains of the genes using the BnaMYB protein sequences, increasing the maximum motif count to 10, the maximum motif amino acid count to 20, and the minimum motif width to 6, and leaving all other variables at their normal values. Finally, the similar motifs of BnaMYB proteins were viewed using TBtools software.

Predicted 3D Structure of R2R3-MYB Proteins
Thirty-five BnaMYB protein sequences were entered into the DNAMAN8.0 program for triple sequence comparison. We next predicted the 3D structure of the proteins using the internet tool Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index, accessed on 25 April 2022).

Analysis of Collinearity within B. napus and Related Species
McScanx analysis was used to analyze the BnaMYB genes' intra-species collinearity in B. napus and Circos software (v0.  was used to visualize the associations. Additionally, the McScanX software (Python version) was used to visualize the collinearity.

Analysis of the Synteny of BnMYB Genes
The CDS sequences of 35 BnaMYB genes were input into DNAMAN8.0 for sequence alignment. We selected the CDS sequences of homologous gene pairs, uploaded them to the ALTER website (http://www.sing-group.org/ALTER/, accessed on 28 January 2023), and outputted the results in ALN format. We converted ALN format to AXT format with AXT Converter and used KaKs Calculator 2.0 software to calculate the value of Ka/KS.

Cis-Acting Elements and Functional Annotation
The publicly available online whole-genome information on B. napus (http://cbi.hzau. edu.cn/bnapus, accessed on 28 April 2022) provided 1500 bp of the upstream sequences of the BnaMYB genes. The cis-acting components were extracted using the online tool Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html, accessed on 29 April 2022) and then visualized using the online tool DSGS.

Spatial and Temporal Expression Patterns of R2R3-MYB Genes
The BnTIR: Brassica napus genomic resource is a website (http://yanglab.hzau.edu.cn, accessed on 2 May 2022) [66]. The stems, roots, siliques, cotyledons, sepals, seeds, buds, leaves, and petals were among the tissues for which we obtained RNA-seq data. To create the heat map of expression, the data were entered into the Heatmapper program (http://www.heatmapper.ca, accessed on 2 May 2022).

Plant Materials and Treatment Methods
In this study, the rapeseed genotype "ZS11", a typical cultivated variety, was used for the stress treatments. The seeds of the ZS11 genotype were furnished by OCRI, CAAS, China. Before the stress treatments, some seeds were randomly selected from the same batch of seeds to determine the germination rate. The seeds with a 100% germination rate were considered to be vigorous seeds. The vigorous seeds were carefully chosen and sterilized with a 10% hypochlorous acid solution for 5 min. The seeds were grown on water-saturated filter paper in a chamber (25 • C day and night, and a 16 h/8 h light/dark cycle) until the plants grew to the five-leaf stage. When the plants had grown to the fiveleaf stage, we stopped watering to impose drought stress. Samples from the stem were collected 0, 1, 2, 3, 4, 5, 6 and 7 days after the initiation of the drought treatment [67,68]. Moreover, three biological replicates of each sample were collected. The gathered stems were immediately put into liquid nitrogen and kept in a freezer for later use at −80 • C.

RNA Extraction and RT-PCR Analysis
Using a carbohydrate phenolic total RNA extraction kit, the total RNA was isolated from leaves that had undergone various drought treatments (Tiangen Biochemical Technology Co., Ltd.). An ultramicroscopic spectrophotometer was used to measure the quantity and quality of the RNA (Thermo Fisher, Waltham, MA, USA, Nanodrop One). As samples for subsequent RT-qPCR experiments, we synthesized cDNA using a reverse transcription kit and diluted it 100 times with ddH 2 O. Specific primers were created using the online qPCR Primer Database (https://biodb.swu.edu.cn/qprimerdb/, accessed on 27 May 2022) based on the coding sequences of the BnaMYB genes. The specific primers were amplified, and the primers with a ct value of no more than 30, a normal amplification curve, and no obvious impurity peak in the dissolution curve were selected for subsequent experiments. The quantitative experiment was conducted in real-time using SYBR Premix Ex TaqTM (TaKaRa). Three separate replicates were collected for this experiment, and the samples that had not been subjected to drought were used as standards. The 2 −∆∆CT technique is often used in investigations of the relative expression of genes [69]. The graphs were developed using GraphPad Prism 9.0.0 software.

Conclusions
In total, 35 BnaMYB genes were found in B. napus, dispersed unevenly across 19 chromosomes. We analyzed how the MYB proteins are related, their gene structure, their conserved motifs, and their three-dimensional structures. These genes were divided into three subfamilies, each of which had reasonably traditional gene architectures and patterns. The BnaMYBs promoter regions also contained cis-acting hormones and abiotic stress response components, along with drought-responsive genes. There were 48 pairs of large-segment repetitive genes discovered in B. napus through the analysis of collinearity. The R2R3-MYB genes of B. napus had undergone polyploidization and various degrees of loss and expansion, according to comparative genomic research. We also looked at the gene expression patterns of BnaMYB in various rapeseed tissues, and the results showed that the members of the BnaMYB gene family were crucial at different phases of the development of B. napus. In addition, we examined the expression patterns of 10 BnaMYB genes under drought. Overall, by carefully examining the conservation and divergence of the BnaMYB gene family's activities, our work lays the biological groundwork for the subsequent functional discovery of R2R3-MYB genes in cruciferous plants.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12040886/s1, Figure S1: Multiple sequence alignment of BnaMYB proteins; Figure S2: Three-dimensional structure prediction of Myb_DNA-binding domain of BnaMYB proteins. The images are from dark blue to dark red, indicating from the N end to the C end; Figure S3: CDS sequence alignment of 35 BnaMYB genes; Table S1: The position of genes in chromosomes;

Conflicts of Interest:
The authors declare no conflict of interest.